MiR-433 mediates ERRγ-suppressed osteoblast differentiation via direct targeting to Runx2 mRNA in C3H10T1/2 cells.

AIMS: MicroRNAs (miRNA) are involved in various biological processes including cellular differentiation. However, the role of miR-433 in osteoblast differentiation remains poorly understood. The objective of this study was to investigate the effect of miR-433 on BMP2-induced osteoblast differentiation. MAIN
METHODS: The expression of mature miR-433 in cells was detected by real-time PCR. RT-PCR or real-time PCR was used to confirm the expression of osteogenic genes. For the activation or inhibition of miR-433 expression, we used a precursor form of miR-433 or anti-miR-433. Functional activity of miR-433 and Runx2 was evaluated by promoter study. Osteoblast differentiation was evaluated by analyzing alkaline phosphatase (ALP) activity. KEY FINDING: ERRγ increased miR-433 expression in the mesenchymal stem cell lineage C3H10T1/2. During the BMP2-induction of osteoblastic differentiation of C3H10T1/2, ERRγ and miR433 expression decreased. In addition, during the osteoblastic differentiation, overexpression of ERRγ or miR-433 inhibited the expression of osteogenic marker genes such as Runx2 and ALP. A computer-based prediction algorithm led to the identification of three miR-433 binding sites [S1 (114-145 bp), S2 (3735-3766 bp) and S3 (3828-3860 bp)] on the 3'-UTR of Runx2 mRNA. Furthermore, miR-433 directly targeted S1 and S2, and decreased the level of Runx2 transcript. In addition, miR-433 inhibited BMP2-induced 6×OSE-Luc activities. Anti-miR-433 recovered ERRγ-suppressed Runx2 expression and ALP activity. SIGNIFICANCE: These results demonstrated that miR-433 suppressed BMP2-indcued osteoblast differentiation by decreasing the level of Runx2 transcript.